Most interactors of protein phosphatase-1 (PP1) contain a variant of a so-called "RVXF" sequence that binds to a hydrophobic groove of the catalytic subunit. A combination of sequence alignments and site-directed mutagenesis has enabled us to further define the consensus sequence for this degenerate motif as [RK]-X 0 -1 -[VI]-{P}-[FW], where X denotes any residue and {P} any residue except Pro. Naturally occurring RVXF sequences differ in their affinity for PP1, and we show by swapping experiments that this binding affinity is an important determinant of the inhibitory potency of the regulators NIPP1 and inhibitor-1. Also, inhibition by NIPP1-(143-224) was retained when the RVXF motif (plus the preceding Ser) was swapped for either of two unrelated PP1-binding sequences from human inhibitor-2, i.e. KGILK or RKLHY. Conversely, the KGILK motif of inhibitor-2 could be functionally replaced by the RVXF motif of NIPP1. Our data provide additional evidence for the view that the RVXF and KGILK motifs function as anchors for PP1 and thereby promote the interaction of secondary binding sites that determine the activity and substrate specificity of the enzyme.The ubiquitous protein serine/threonine phosphatases of type 1 (PP1) 1 and type 2A (PP2A) interact with dozens of different polypeptides that function as substrates, inhibitors, chaperones, anchoring/scaffolding proteins, or substrate-specifiers and are often multifunctional (1-3). For example, the glycogen-associated G-subunits not only target PP1 to the glycogen particles but also increase the specific activity of PP1 toward the substrate glycogen synthase. Similarly, protein kinase Nek2 is a substrate for associated PP1 and targets the centrosomal protein C-Nap1 for dephosphorylation by PP1. In addition, Nek2 mediates cell cycle-dependent control of centrosomal PP1. The promiscuity of PP1 and PP2A in their interaction with other polypeptides accounts for the presence of these enzymes in a large variety of different holoenzymes. The sharing of catalytic subunits between holoenzymes also explains why higher eukaryotes can manage with 15 times fewer protein serine/threonine phosphatases than protein serine/threonine kinases (4).Mammalian genomes contain three genes that encode isoforms of PP1 (1, 3). These isoforms (35-38 kDa) are about 90% identical, and the differences are mainly concentrated in the extremities. Although some PP1 interactors such as the neurabins (5, 6) interact with PP1 in an isoform-specific manner, most interactors do not discriminate between PP1 isoforms, implying that the major interactor binding sites reside in the catalytic core, i.e. the central three-quarters of the protein. The surface of the catalytic core is too small to harbor specific binding sites for each of the 65 known mammalian interactors. The available evidence rather suggests that PP1 interactors compete for a limited number of common or overlapping binding sites (discussed in Ref.

Background A hallmark of heart failure is impaired cytoplasmic Ca2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca2+ handling – via altered excitation-transcription coupling – contribute to the development and progression of heart failure. Methods and Results Using tissue and isolated cardiomyocytes from non-failing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca2+ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment there was also reduced expression of Ca2+ pumps and ryanodine receptors, and increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca2+ transporters. These changes were associated with altered nucleoplasmic Ca2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca2+ levels with diminished and prolonged nuclear Ca2+ transients and slowed intranuclear Ca2+ diffusion. Altered nucleoplasmic Ca2+ levels were translated to higher activation of nuclear Ca2+/calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca2+ changes that are typical of heart failure. Conclusions During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca2+ regulation may, therefore, be a novel therapeutic approach for preventing adverse cardiac remodeling.

The elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.